Dynamical View of Energy Coupling Mechanisms in Active Membrane Transporters
Emad Tajkhorshid
Computational Structural Biology and Molecular Biophysics www.csbmb.beckman.illinois.edu
Department of Biochemistry
Center for Biophysics and Computational Biology
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
Proton-Driven Sugar
Transport in LacY
25th Molecular Modeling Workshop
April 2011, Erlangen, Germany
ADP/ATP Exchange in
Mitochondria (AAC)
Mechanically(?) Driven
Transporters of the Outer
Membrane
ATP-Driven Transport in
ABC Transporters
Na + -Driven
Neurotransmitter Uptake
Glutamate Transporter

Solving the Newtonian equations of motion for all particles at every time step
Major limitations:
§ Time scale / sampling
§ Force field approximations
SPEED
LIMIT
Major advantage:
1 fs
§ Unparalleled spatial and temporal resolutions, simultaneously

Access to More Computational Power
HP 735 cluster
12 processors
(1993)
?
Blue Waters (UIUC)
200,000+ processors (2012)
SGI Origin 2000
128 processors (1997)
PSC LeMieux AlphaServer SC
3000 processors (2002)
Ranger/Kraken
~60,000 processors (2007)
Anton/DESHAW/PSC
512 processors (2010)

• Active transport is coupled to an energy source in the cell
• Transporters Function on μ s and longer time scales
• Protein conformational changes of various forms and magnitudes coupled to step-wise vectorial translocation of the substrate and co-transported materials
• The sequence of molecular events is largely unknown

In situ Molecular Dynamics Simulations
Atom count: 100-500k
~10 ns/day on 128-1024 processors
100-500 ns for each system

Hard to define the number of (sub)states involved?

Amara and Fontana, Neurochemisty International
41:313-318 (2002)
Glutamate Transporter (Glt ph
)
Yernool, Boudker, Jin, Gouaux. Nature, 431: 811–818, 2004 .

Sequence and Coupling of Events in an Ion-Coupled Transporter
HP1
HP2
Z. Huang and E. Tajkhorshid, Biophysical Journal 2008
Glutamate Transporter (Glt ph
)
Yernool, Boudker, Jin, Gouaux. Nature, 431: 811–818, 2004 .

w/ substrate w/o substrate
HP2 t
HP2
0
HP2 t HP2 0
HP2
Z. Huang and E. Tajkhorshid, Biophysical Journal 2008
HP1

Ion-Substrate Coupling on the Extracellular Side w/o substrate w/ substrate
Substrate binding forms the Na2 binding site
Na2 seals the binding site
Z. Huang and E. Tajkhorshid, Biophysical Journal 2008

ph
Reyes, et al., Nature 2009

Different Modes of Cytoplasmic (HP1) and
Extracellular (HP2) Gating periplasm
Leighton, et al., JBC (2006)
Coupled to substrate release
Na1-controlled inward -facing gate outward -facing gate
Substrate-controlled
Coupled to Na2 binding cytoplasm

Na1 Dependence of the Cytoplasmic Gate
Na1
Inward-facing, occluded with Na1/substrate
Occluded to open transition after Na1 release

vSGLT: A Secondary Membrane Transporter in the Occluded Inward-Facing State
Na + was modeled based on LeuT Faham et al., Science, 810-814, 2008

Spontaneous Na + Unbinding in Multiple Simulations
D189
• Several independent simulations, all resulting in Na + unbinding
• The crystal structure is not an occluded state, rather an open inward-facing state.
J. Li and E. Tajkhorshid, Biophysical Journal 2009
D189

Comparison of the Na + Binding Sites in vSGLT, LeuT and Mhp1 vSGLT: yellow
LeuT: pink
Mhp1: orange
vSGLT: open state
LeuT,Mhp1: occluded state
J. Li and E. Tajkhorshid, Biophysical Journal 2009

Artificially Recovering the Occluded State

Substrate-bound state substrate-free state ?

Early Stage of Substrate Release Captured by Free MD

Transporter Dogma: Alternating Access
Mechanism
Transporters switch the substrate access between the two sides.
The central binding site should never be exposed to both sides simultaneously .

TMDs
NBDs
TMDs
NBDs
TMDs
NBDs
• Architecture
–2 NBDs
• Conserved domains
• ATPase activity
–2 TMDs
• Diverse sequence and structure
• Substrate transport
–1 BP
• ABC importers only
• Substrate recognition and binding

Nucleotide-Dependent State of NBDs
Lu et. al.
, PNAS , (2005)

• MalK dimer ( 1Q12.PDB
)
• Placing Mg 2+
• Solvate ( 80,000 atoms )
• Equilibrium MD - 75 ns
• 4 simulation systems
– ATP / ATP
– ADP-P i
/ ATP
– ATP / ADP-P i
– ADP-P i
/ ADP-P i
1 or 2 ATP hydrolysis?
Hydrolysis or release of products?

ADP/Pi-Bound
ATP-Bound
P. Wen and E. Tajkhorshid, Biophysical Journal 2008

ATP/ATP 2 Hydrolysis
1 hydrolysis - top 1 hydrolysis - bottom
P. Wen and E. Tajkhorshid, Biophysical Journal 2008

A
LSGGQ
Walker A
LSGGQ
B
Pi
A
L
S
G
G
Q
Q
G
G
S
L
B
Pi rib
A
A
L
S
G
G
Q
Q
G
G
S
L
A B rib
A
Walker A
S
D
D
S
B
P. Wen and E. Tajkhorshid, Biophysical Journal 2008

(B)
Lys42
ATP bound
Gly137
Gly136
Ser135
Ala133
Meta-stable ADP state
P. Wen and ET, Biophys. J. , 2008.

• Two subdomains
– RecA-like subdomain
• Majority of ATP binding site
• Walker A motif
– Helical subdomain
• Complimentary to ATP binding
• Signature “LSGGQ” motif
• Two nucleotide binding sites
– Both at the dimer interface
– “Nucleotide-sandwiched” dimer
PDB:1Q12
ATP binding à dimerization
Hydrolysis à dimer opening
Why?

in the maltose transporter
MalEFGK, ATP-bound
~320,000 atoms averaged between t = 70-80 ns
+500 mV isosurface
MalEFGK, Nucleotide-Free
~320,000 atoms averaged between t = 70-80 ns

in the molybdate/tungstate transporter
ModABC Nucleotide-Free
~220,000 atoms averaged between t = 0-10 ns
+500 mV isosurface
ModABC, ADP docked
~220,000 atoms averaged between t = 0-10 ns

in other ABC transporters
MsbA ATP-bound
~200,000 atoms averaged between t = 0-10 ns
+500 mV isosurface
BtuCDF, Nucleotide-Free
~220,000 atoms averaged between t = 0-10 ns

R139
R146
R141

Key Role of Buried Charges in NBD Dimerization
R141
R139
R146
Wildtype Arg139 neutralized
MalK dimers
All ATP Bound t = 50 ns
Arg141 neutralized
Arg146 neutralized

Recovery of NBD Dimerization in the Mutants
Arg 139 Arg 140 -Gln 141 Gln 139 -Arg 140 -Arg 141
(wt = Arg 139 -Gln 140 -Arg 141 )

Recovery of NBD Dimerization in the Mutants
Conclusion: Buried positive charges in the helical subdomains are essential for NBD dimerization

•Three factors determine the
NBD dimerization
– Binding
• Walker A motif binds ATP
– Attracting
• Positive charged residues in the helical subdomain
• Optimal: 2 positive charges near the LSGGQ motif
• Location, location, location!
– Locking
• Network of hydrogen bonds between the ATP and the LSGGQ motif

Unusually Strong Electrostatic Potential
Average electrostatic potential of AAC
• Very strong (~1.4V) positive potential at the AAC basin provides the driving force for ADP binding.
Y. Wang and E. Tajkhorshid, PNAS 2008

Commonality of Electrostatic Features in
MCF Members
• Almost all MCF members are strongly charged (positive).
• Most substrates of MCFs are negatively charged.
– Substrate recruitment
– Anchoring the proteins into the negatively charged inner mitochondrial membrane.
Y. Wang and E. Tajkhorshid, PNAS 2009

GlpT
N-terminal domain (6 TMH)
C-terminal domain (6 TMH)
Putative binding site
R269
H165
R45
Inter-domain loop
(not resolved in the crystal) phosphate/organic phosphate antiporter
Huang, et al., Science 301 , 616-620 (2003)

Ch. Law, G. Enkavi, D.-N. Wang and E.
Tajkhorshid, Biophysical Journal 2009
G. Enkavi and E. Tajkhorshid, Biochemistry 2010

Phosphate Binding Site
Glycero-phosphate
Binding Site
Ch. Law, G. Enkavi, D.-N. Wang and E.
Tajkhorshid, Biophysical Journal 2009 .

Capturing the Initial Steps of the
Rocker-Switch Mechanism
Substrate induced straightening of
H5 H11
Cytoplasmic View
H5
H11
N-term C-term
G. Enkavi and E. Tajkhorshid, Biochemistry 2010

Computational Structural
Biology and Molecular
Biophysics Group (CSBMB) csbmb.beckman.illinois.edu
Anton